New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.


Introduction
In vertebrates, the undifferentiated gonads rely on genetic and environmental sex determination (GSD and ESD) to determine their differentiation along male or female differentiation pathways [1,2].In teleosts, the GSD are complex due to diversity within the species [3], and diverse master sex determination genes, including dmrt1, dmrt1bY (DMY), anti-Müllerian hormone Y (amhy), gsdfY, etc [4][5][6][7][8][9].Additionally, some teleosts may ultimately tip the bipotential gonads towards the male or female fate in response to a continuum of genetic and environmental factors [10,11].The domesticated experimental strains of zebrafish have lost their natural sex determinants, and lack a single strong genetic determinant [12].
Gonadal sex differentiation is further influenced by sex steroid hormones, especially androgens and estrogens, which are produced by the different steroidogenic lineages in the somatic cells of the testes or ovaries [13].Genes encoding enzymes involved in sex steroid synthesis are differentially expressed during gonadal differentiation [3].Cyp19a1a, an aromatase, converts testosterone to the estrogen 17β-estradiol (E2).Estrogen is known to be essential for ovary differentiation and maintenance, as indicated by the all-male phenotype observed in cyp19a1a-/deficient zebrafish [14][15][16].Additional depletion of doublesex and mab-3 related transcription factor 1 (dmrt1), which drives male differentiation and maintains testis development, in cyp19a1a-/-deficient zebrafish resulted in partial ovary differentiation [17][18][19].Cyp17a1, a cytochrome P450 enzyme with 17-alpha-hydroxylase and C17,20-lyase activities, is the key enzyme in the production of androgen and estrogen in animals [20].Depletion of cyp17a1 in zebrafish leads to all-testis differentiation, loss of male-typical secondary sex characteristics and mating behavior, due to their impaired androgen production [21][22][23].Similarly phenotypes are also observed in cyp17a1-/-common carp and scl (sex-character-less, P450c17) mutant medaka, irrespective of the individuals' sex-determining genotypes (XY or XX) [24,25].The increased progestin signaling seen in cyp17a1-/-fish was found to be responsible for proper testis development and spermatogenesis, which is dependent on the nuclear Progestin receptor (nPgr).It has also been shown that the increased endogenous progestin signaling does not have any effect on the sexual differentiation of the cyp17a1-/-zebrafish [23].Theoretically, the double knockout of cyp17a1 and dmrt1 could further help to elucidate the mechanism underlying all-testis differentiation in cyp17a1-/-zebrafish with impaired endogenous androgen and estrogen signaling.
Zebrafish gonads initially form an ovary-like structure (called a "bipotential juvenile ovary"), which then develops into either into the ovaries in females or testes in males [26,27].Gonadal differentiation follows oocyte apoptosis in the bipotential juvenile ovary during a critical window of time (20-30 days post-fertilization, dpf) that lasts for several days [27][28][29].In zebrafish, the number of germ cells influences gonadal sex differentiation [30,31].The complete loss of germ cells in dnd morphants leads to an all-male, sterile zebrafish phenotype [28,32,33].It has been suggested that adult females can undergo sex reversal from female to male when oocytes in the mature ovary are depleted, as observed in nanos3-null mutants or ziwi-CFP-NTR transgenic zebrafish at age of 5 months post-fertilization (mpf) [30]; Apparently, a sufficient number of germ cells is essential for the female differentiation in zebrafish [31,32].Therefore, specific levels of oocyte-derived signals are thought to act on somatic cells in the gonads to promote and maintain ovary differentiation in zebrafish.
Fancl plays an important role in several biological processes, including DNA damage and apoptosis.In humans, fancl mutation leads to Fanconi Anemia (FA), which is a disease characterized by failure of bone marrow production, risk of developing cancer, hypogonadism, and impaired fertility [34].Mutations in the FA pathway genes disrupt the repair of DNA damage caused by DNA inter-strand crosslinking [35].Among FA genes, fancc, fancg, fanca, fancd1 (brac2), and fancd2 are known to cause hypogonadism, impaired gametogenesis, and infertility [36].In zebrafish, a complete female-to-male sex reversal was observed for 12 of the 17 FA mutants, including fancd1 and fancj homozygous knockout fish that were infertile [35,37].Fancl transcripts begin to be observed in immature gonads at 17 and 23 dpf; their expression levels increase in developing germ cells at 26 dpf and persist to increase in developing oocytes and spermatocytes at 33 and 37 dpf [38].The insufficient number of surviving oocytes caused by fancl depletion masculinizes the gonads in mutant zebrafish [37].One factor known to mediate apoptosis in the developing gonad is Tp53 [39].Specifically for male differentiation, Tp53-mediated apoptosis is required for the all-male phenotype in brac2-/-or fancl-/-zebrafish, as the all-male phenotype caused by increased apoptosis in the gonads could be rescued by tp53 mutation [37,38].However, tp53 depletion can only partially rescue the sex reversal phenotypes exhibited by fancd1, fancl, fancr and fancp mutant zebrafish [35,37,38,40].These results suggest a possible link between certain FA genes and Tp53, with elusive mechanisms determining gonadal differentiation.
In this study, all-female differentiation was observed in cyp17a1-/-;dmrt1-/-fish accompanied by significantly up-regulated fancl expression.Dmrt1 and androgen signaling probably stabilize Tp53 via inhibiting the transcription of fancl, which interacts with Tp53 and activates its K48-linked ubiquitination.Assuming that similar regulatory relationships exist in the germline and gonads, our results provide mechanistic insight into a novel regulatory function of the interactive germline signals and gonadal somatic signals in teleost gonadal sex determination.
In support of this hypothesis, up-regulation of fancl was observed in the cyp17a1-/-; dmrt1-/-fish compared to the control females as verified by qPCR at 17 and 23 dpf (Fig 2A and 2B).Moreover, significantly up-regulated fancl expression was observed in dmrt1-/-fish at 17 dpf, while fancl up-regulation was not significant in dmrt1-/-fish at 23 dpf (Fig 2A and 2B).As cyp17a1-/-;dmrt1-/-fish all developed as females, we analyzed the expression level of fancl by in situ hybridization in presumptive ovaries from control fish and cyp17a1-/-;dmrt1-/-fish at 25 dpf.Compared to the moderate expression of fancl in the presumptive ovary of control fish, that of cyp17a1-/-;dmrt1-/-fish was significantly higher (Fig 2C -2E).In situ hybridization using the sense probe of fancl was also performed on cryosections of presumptive ovaries and as expected, no signals were detected (S2A and S2B Fig).
Comparative transcriptomic analyses were performed between ovaries from cyp17a1-/-; dmrt1-/-fish and cyp17a1+/+;dmrt1+/+ female control siblings at 80 dpf.Compared to the ovaries from control fish, those from cyp17a1-/-;dmrt1-/-females exhibited significant expression level alterations of 424 genes (Fig 2F ), with 238 genes being up-regulated and 186 genes being down regulated.The most enriched KEGG pathways in cyp17a1-/-;dmrt1-/-fish were significantly up-regulated as shown in Fig 2G .The top enriched pathways were related to intestinal immune network for IgA production, steroid hormone biosynthesis, Toll-like receptor signaling, Notch signaling and FA signaling (fancl).The up-regulated fancl expression in cyp17a1-/-;dmrt1-/-fish ovaries at 80 dpf was further verified by qPCR (Fig 2H).These differentially expressed genes may have important functions in ovary differentiation of cyp17a1-/-; dmrt1-/-fish, although it still could not be excluded that their high expression was caused by their expression in primary oocytes or lower abundance in growing and mature oocytes.
To inspect whether Dmrt1 and androgen signaling could transcriptionally regulate fancl expression, a 2.5 kb region upstream of the zebrafish fancl transcription site was cloned into pGL3-basic vector.Both Dmrt1 and dihydrotestosterone (DHT)/Androgen receptor (Ar) inhibit the relative luciferase activity driven by fancl promoter, and their combinational treatment resulted in the largest inhibitory effect in vitro (Fig 2I).
We observed that the testes of dmrt1-/-;fancl-/-fish were also hypoplastic and lack germ cells similar to dmrt1-/-fish, compared to the control fish (Fig 3Q -3C1).The results highlighting the antagonistic role of Fancl and Dmrt1 in determining gonadal sex, not only suggest that Fancl is required for ovary differentiation in dmrt1-/-;fancl-/-fish, but also imply the existence of other Dmrt1 targets required for testis development, as the testis is impaired in dmrt1-/-; fancl-/-fish.

Zebrafish Fancl interacts with Tp53 in vitro
Fancl plays an important role in the survival of developing oocytes during meiosis, and Tp53-mediated germ cell apoptosis induces sex reversal in fancl mutant zebrafish [38].Given the higher expression of fancl in the gonadal tissue of cyp17a1-/-;dmrt1-/-fish, which is an allovary differentiation context, we postulated that Fancl may play a role in ovary differentiation by interacting with the Tp53 signaling cascade.To test this hypothesis, a co-immunoprecipitation assay was performed in HEK293T cells.Myc-tagged Fancl and Flag-tagged Tp53 plasmids were transfected into HEK293T cells.The interaction between exogenously Flag-tagged Tp53 and Myc-tagged Fancl was observed by a reciprocal Co-immunoprecipitation (Co-IP) experiment (Fig 4A).Domain mapping of the interaction between Fancl and Tp53 indicated that the DNA-binding domain (DBD) of Tp53 is required for their interaction (Fig 4B and 4C).The multiple domains, WD-repeat domain (WDR) and C-terminal domain (CTD), of Fancl are required for their interaction, rather than single functional domain ( Figs 4D, 4E, S4A and S4B).

Discussion
The key functions of estradiol in zebrafish gonadal sex determination have been extensively documented [14,15,21].In cyp17a1-/-zebrafish and common carp, it would be interesting to explore the mechanisms underlying the all-testis differentiation and successful spermatogenesis [21,25].Augmentation of progestin signaling has been proposed to be responsible for the proper testis organization and spermatogenesis in cyp17a1-/-;ar-/-zebrafish.However, additional depletion of npgr in cyp17a1-/-;ar-/-fish leads to the phenotypes of all-testis differentiation with impaired spermatogenesis in cyp17a1-/-;ar-/-;npgr-/-zebrafish [23].This result suggests progestin is important for normal organization of the testis and spermatogenesis, but not for determination of testis fate [23].
In zebrafish, fancl was up-regulated in the presumptive ovaries as compared with the presumptive testes [41].In our analyses, the increased expression of fancl in presumptive ovaries of cyp17a-/-;dmrt1-/-fish at 17, 23 and 25 dpf compared to that in control females was positively correlated with their female biased sex ratio.Indeed, we also observed a moderate upregulation of fancl in dmrt1-/-fish at 17-23 dpf; however, it is not as significant as in cyp17a-/-;dmrt1-/-fish.This is unlikely to be driven by differences in the stages of oocytes present in the ovaries in our different mutant contexts, as the dmrt1-/-female zebrafish is fertile and develops normally [42].The observed synergy between Dmrt1 and androgen signaling in inhibiting fancl transcription in luciferase reporter assays suggests that elevated fancl in cyp17a1-/-; dmrt1-/-fish may result from loss of these synergistic repressors.
Disruption of fancl is known to cause masculinized gonads and testis differentiation due to increased germ cell apoptosis compromises oocyte survival, which could be rescued by tp53depletion [38].Fancl is known as a ubiquitin ligase [34].Our in vitro results indicate that zebrafish Fancl interacts with Tp53 to promote its degradation through K48-linked polyubiquitination.Assuming that similar regulatory relationships exist in the germline and gonads in vivo, these results provide new insights into the regulatory network involved in Fancl functions for ovary differentiation, which may protect germ cells from apoptosis induced by Tp53 signaling in zebrafish.Notably, increased apoptosis and decreased Vasa-positive germ cells were observed with dmrt1 deficiency previously [42].Additional depletion of fancl resulted in alltestes differentiation in cyp17a1-/-;dmrt1-/-fish, due to impaired ovary differentiation.Therefore, it could be concluded that the Fancl-mediated germ cell survival is determinant in gonadal differentiation in cyp17a1-/-;dmrt1-/-zebrafish.
The severely hypoplastic testes observed in dmrt1-/-;fancl-/-fish and cyp17a1-/-;dmrt1-/-; fancl-/-fish are consistent with our previous view that Dmrt1 is required for the maintenance of male germ cells [42].This could be interpreted that additional fancl depletion promotes testis differentiation in dmrt1-/-fish and cyp17a1-/-;dmrt1-/-fish, but their male germ cell development is dysregulated due to Dmrt1 deficiency.Similar observations have been reported with zebrafish whole testis differentiation upon depletion of RNA-binding protein of multiple splice forms 2 (rbmps2), a critical germline-expressed factor for female sex differentiation.The severe hypoplastic testes were observed in dmrt1-/-;rbmps2a-/-;rbmps2b-/-zebrafish [18].Significantly, depletion of genes related to germ cell development and survival in fish results in the same phenotype of the lost germ cells as they attempt to embark on the male fate but suffer from the lack of Dmrt1 [18].
Loss of tp53 can restore ovarian development in fancd1(brca), fancl, fancp and fancr mutant fish [35].In contrast, introducing tp53 mutation did not restore ovary differentiation in dazl, figla, rbm46, vasa and rbpms2 mutants, which are key factors involved in germ cell survival, meiosis and differentiation [46][47][48][49][50].We also observed that depletion of tp53 (IHB136, China Zebrafish Resource Center) did not affect sexual differentiation in zebrafish and could not restore ovary differentiation in cyp17a1-/-zebrafish.This suggests that germ cell loss is not exclusively mediated by apoptosis via Tp53.Further studies, such as generating and analyzing dmrt1-/-;fancl-/-;tp53-/-zebrafish, are needed to investigate the mechanisms of Dmrt1 on Fancl/Tp53 signaling on the link between sex fate determination and germ cell survival.
Increased progestin signaling maintains proper testis organization and spermatogenesis in cyp17a1-/-;ar-/-zebrafish, suggesting a dispensable role for androgen in testis organization and spermatogenesis under certain circumstances [23].In contrast, only PG and PV stage follicles were observed in cyp17a1-/-;dmrt1-/-zebrafish at 90 dpf.Together with a similar follicle status observed in cyp19a1a-/-;dmrt1-/-zebrafish, the arrested follicle development from PV to EV transition with impaired vitellogenesis could be attributed to impaired estrogen synthesis when either cyp19a1a or cyp17a1 was depleted in these mutants [17,18].The vitellogenic (EV+) follicles were observed in cyp17a1-/-;dmrt1-/-fish after 17β-estradiol administration from 80 to 110 dpf, supporting the essential function of 17β-estradiol in regulating folliculogenesis via vitellogenesis.Ovarian follicles with failed yolk accumulation were also observed in cyp19a1a-/-female medaka [51].Taken together, the endogenous estrogens synthesized in zebrafish and medaka are dispensable for ovarian differentiation, but indispensable for ovarian development and oocyte maturation.On the other hand, although cyp17a1 deficiency leads to elevated progestin levels (in cyp17a1-/-fish) [23], it is not sufficient to provide an adequate gonadal steroid hormone environment for folliculogenesis in cyp17a1-/-;dmrt1-/-zebrafish.
Determination of the fate of gonadal supporting cells in mammals plays a critical role in gonadal sex determination, as Sry acts spatiotemporally to switch supporting cells from the female to the male pathway [52].Both in XX and XY mice, depletion for CYP17A1 exclusively causes phenotypically female appearance (external genital phenotype), abnormal inner genitalia development and infertile phenotypes [53].Compared to the phenotype observed in CYP17A1-null mice, all-testis differentiation and proper spermatogenesis were observed in cyp17a1-/-zebrafish and common carp [21,25].These results demonstrate the evolutionary plasticity of sex determination and gonadal development in vertebrates.Of course, further studies of our proposed regulatory mechanism are needed in other fish species or mammals, which, we believe, would undoubtedly broaden the knowledge underlying sex determination in teleosts.

Ethics statement
All fish experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, and were approved by the Institute of Hydrobiology, Chinese Academy of Sciences (Approval ID: IHB 2013724).

Zebrafish maintenance
Zebrafish (Danio rerio) were maintained as previously described [54].Briefly, the fish were kept in a circulated water system and maintained under standard laboratory conditions at 28.5˚C with a light/dark cycle of 14/10 hours; the Fish were fed twice daily with freshly hatched brine shrimp.

Histological analysis
Hematoxylin and eosin staining was performed as previously described [21].Dissected gonads were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature.Fixed samples were dehydrated, infiltrated, and embedded in paraffin for sectioning.Sections

In situ hybridization
The in situ hybridization on cryosections of presumptive ovaries were performed as previous described [56].Sense and anti-sense digoxigenin-labeled cRNAs of fancl were synthesized and used in this study.The cDNA fragment of 786 nt containing the PHD domain of fancl was used to synthesize probe as previously described [38].The in situ hybridization were photographed using a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan).Staining intensity in germ cells were quantified by analyzing the gray values using Image J software (National Institutes of Health, Bethesda, MD, USA).There were three independent replicates for the fish of each genotype.

Isolation and staging of ovarian follicles
The staging system adopted for ovarian follicles was conducted based on the original definition of Selman et al. [57] as modified by the researchers [17,[58][59][60][61]. Ovaries, which were dissected out from females of each genotype after anesthetization, were placed in a 60 mm culture dish containing 60% L-15 medium (Gibco, Carlsbad, CA, United States).The follicles of different stages were manually isolated and divided into five stages according to their size and vitellogenic stage: primary growth stage (~0.1 mm), previtellogenic stage (cortical alveolus, ~0.3 mm), early vitellogenic stage (~0.4 mm), midvitellogenic stage (~0.5 mm), and full grown but immature stage (~0.65 mm).

Serum estradiol measurement
The concentrations of serum estradiol in fish at 3 mpf were measured using commercial ELISA kit (582251, Cayman Chemical Company, Ann Arbor, MI) as previously described [21].There were four independent replicates for the fish of each genotype.

RNA extraction and quantitative real-time PCR (qPCR)
Total RNA was extracted from zebrafish using TRIzol reagent (15596-026; Invitrogen, Carlsbad, CA, United States) following a previously described standard protocol [21].For zebrafish at 17 and 23 dpf, truncated zebrafish bodies, which contained the gonads, were used for RNA extraction.We synthesized cDNA using One-Step gDNA Removal and cDNA Synthesis SuperMix (AE311-02; Transgen Biotech, Beijing, China).qPCR was performed using SYBR Green Real-Time PCR Mix (AQ131-01; Transgen Biotech) and a real-time PCR system (Bio-Rad, Hercules, CA, United States).The housekeeping gene β-actin was used as endogenous control, and the expression level of fancl was calculated as the fold change relative to β-actin [62].The primers used for qPCR are listed in Table 1.The cyp17a1+/+;dmrt1+/+ female fish of the cyp17a1-/-;dmrt1-/-siblings served as control.The comparison was performed between the fish of different genotypes.

Transcriptome analysis
At the indicated time points, total RNA from the dissected ovaries was extracted using TRIzol reagent.RNA-seq reads were generated using the Illumina NovaSeq 6000 system.High-quality mRNA reads were mapped to the Danio rerio genome (GRCz11) using HISAT2 (version 2.2.4,http://daehwankimlab.github.io/hisat2/).Differential expression analysis was performed using the DESeq2 package (v1.30.1) with a fold change of two and a p-value cutoff of 0.05.

Plasmid constructions
Zebrafish Fancl, Tp53, Dmrt1 and Ar were cloned into the vectors of pCMV-myc modified pCMV-flag, and pcDNA3.1(+),respectively.For fancl luciferase construction, a 2.5 kb region upstream of the transcription initiation site of zebrafish fancl was cloned into the pGL3-basic plasmid.The primers used for fancl promoter amplification are listed in Table 1.

Luciferase reporter assay
Luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer's instructions (E1910, Promega, Madison, WI, United States).Data were normalized to Renilla luciferase.The relative luciferase activity in transfected cells was detected using a Sirius Luminometer from Berthold Detection Systems.Data were obtained from three independent experiments.

Co-IP analysis
For Co-IP analysis, HEK 293T cells grown to 60% confluency were transfected with a total of 10 μg of the indicated plasmids.At 24 h post-transfection, the medium was carefully removed, and the cells were washed with ice-cold PBS.Then the cells were lysed in 1 mL RIPA buffer at 4˚C on a horizontal shaker for 1 h.Cells lysates were centrifugated at 16000 × g at 4˚C for 20 min, then the supernatant was incubated with anti-Flag (M8823; Sigma-Aldrich, St. Louis, MO, United States) or Myc magnetic beads (88842, Thermo Fisher Scientific, Waltham, MA, United States) overnight at 4˚C.Finally, immunoprecipitates and total cell lysates (TCL) were analyzed by western blotting using the indicated antibodies.

Ubiquitination inhibitor administration
HEK 293T cells were transfected with the intended plasmids.At 24 h post-transfection, the cells were treated with proteasome inhibitor MG132 (10 μg/mL) (S2619; Selleck, Shanghai, China) or dimethyl sulfoxide for 6 h.Total protein from the cells was extracted using RIPA buffer for western blot analysis.

Ubiquitination assay
Transfected HEK 293T cells were washed twice with ice-cold PBS, lysed in buffer A (6 M guanidium-HCl, 0.1 M Na 2 HPO 4 /NaH 2 PO 4 and 10 mM imidazole) and incubated with Ni2 +-NTA beads (Qiagen, Germantown, MD, United States) at 4˚C on a horizontal shaker overnight.The beads were washed sequentially with wash buffer I (mix buffer A and wash buffer II to a ratio of 1 to 4) and wash buffer II (25mM Tris-Cl pH 8.0 and 20mM imidazole) three times.The bound proteins were eluted with wash buffer II and subjected to western blotting.

Statistical analysis
Each experiment was performed in triplicate.Detailed information regarding the number of zebrafish used per experiment is provided for each experiment and corresponding figure.The results are expressed as the mean ± SD.All analyses were performed with the GraphPad Prism 6.0 software program and the differences were assessed using the Student's t-test for paired comparisons and one-way ANOVA, followed by Fisher's LSD test for multiple comparisons.For all statistical comparisons, a p value < 0.05 was used to indicate a statistically significant difference.Significant differences marked with asterisks and letters were analyzed using Student's t-test for paired comparisons, and one-way ANOVA followed by Fisher's LSD test for multiple comparisons, respectively.

Fig 2 .
Fig 2. The cyp17a1-/-;dmrt1-/-fish exhibited increased expression of fancl.(A and B) Relative expression of fancl in control fish, dmrt1-/-fish and cyp17a1-/-;dmrt1-/-fish at 17 dpf and 23 dpf was tested with qPCR.For fish RNA sampling at 17 dpf and 23 dpf, every 5 body trunks of fish collected were mixed into one sample, and 3 samples were examined.(C and D) In situ hybridization was performed on cryosections of presumptive ovaries from control fish and cyp17a1-/-;dmrt1-/-fish at 25 dpf using the antisense probe of fancl.Arrows point to the immature oocytes.(E) Comparison of differentially expressed gene in the ovaries of control fish and cyp17a1-/-;dmrt1-/-fish at 80 dpf.Volcano plot shows genes that were differentially expressed in ovaries between control and cyp17a1-/-;dmrt1-/-fish. (F) Gene set enrichment analysis based on genes differentiated expressed in cyp17a1-/-;dmrt1-/-fish at 80 dpf.The length of the bar represents the false discovery rate (FDR).(G) Relative expression of fancl in the ovaries of control fish and cyp17a1-/-;dmrt1-/-fish at 80 dpf with qPCR.For fish at 80 dpf, every three dissected ovaries were mixed as one sample, and 3 samples were examined.(H) The effect of Dmrt1 and DHT/Ar in regulating the relative luciferase activity driven by fancl promoter.DHT, dihydrotestosterone. ***, p < 0.001.Different letters in the bar chart represent significant differences.https://doi.org/10.1371/journal.pgen.1011170.g002

Fig 4 .
Fig 4. Fancl interacts with Tp53 in HEK293T cells.(A) The interaction of Fancl with Tp53 in HEK293T cells as revealed by the Co-IP assay.Myc-tagged Fancl and Flag-tagged Tp53 were transfected into HEK293T cells, then anti-Flag antibody-conjugated agarose beads were used for immune-precipitation. (B and C) Domain mapping revealed that the DBD domain of Tp53 is required for their interaction.TAD, transactivation domain.DBD, DNA binding domain.TMD, tetramerization domain.(D and E) Domain mapping revealed that the multiple domains, WDR and CTD, of Fancl are required for their interaction.WDR, WD-repeat domain.ULD2, UBC-like domain 2. ULD3, UBClike domain 3. CTD, C-terminal domain.IP, immunoprecipitation.IB, immunoblotting.TCL, total cell lysate.https://doi.org/10.1371/journal.pgen.1011170.g004

Table 1 . Primers used in this study.
μm-thick) were stained with hematoxylin and eosin and visualized under a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan).The oocytes at 50 and 90 dpf are shown with with a scale bar of 50 and 300 μm, respectively.Normal testes are shown with a scale bar of 25 μm, and the hypoplastic testes are shown with a scale bar of 10 μm.